NMR Characterization of Long-Range Order in Intrinsically Disordered Proteins

Abstract

Intrinsically disordered proteins (IDPs) are predicted to represent a significant fraction of the human genome, and the development of meaningful molecular descriptions of these proteins remains a key challenge for contemporary structural biology. In order to describe the conformational behavior of IDPs, a molecular representation of the disordered state based on diverse sources of structural data that often exhibit complex and very different averaging behavior is required. In this study, we propose a combination of paramagnetic relaxation enhancements (PREs) and residual dipolar couplings (RDCs) to define both long-range and local structural features of IDPs in solution. We demonstrate that ASTEROIDS, an ensemble selection algorithm, faithfully reproduces intramolecular contacts, even in the presence of highly diffuse, ill-defined target interactions. We also show that explicit modeling of spin-label mobility significantly improves the reproduction of experimental PRE data, even in the case of highly disordered proteins. Prediction of the effects of transient long-range contacts on RDC profiles reveals that weak intramolecular interactions can induce a severe distortion of the profiles that compromises the description of local conformational sampling if it is not correctly taken into account. We have developed a solution to this problem that involves efficiently combining RDC and PRE data to simultaneously determine long-range and local structure in highly flexible proteins. This combined analysis is shown to be essential for the accurate interpretation of experimental data from R-synuclein, an important IDP involved in human neurodegenerative disease, confirming the presence of long-range order between distant regions in the protein.

Functionally specific binding regions of microtubule-associated protein 2c exhibit distinct conformations and dynamics

“…Both ensembles were joined together to define a starting pool of 84,000 conformers and experimental observables were calculated for each conformer. Chemical shifts were predicted using SPARTA (36), SAXS using CRYSOL (73)(74)(75), and PRE as described previously (76). Local alignment windows of 15 amino acids in length were used to calculate averaged RDCs (77).…”

Introducing Protein Intrinsic Disorder

“…This situation has changed by the advent of methods based on residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs), which yield a high number of well-defined quantitative parameters reporting on long-range interactions (PRE) and global structure (PRE, RDC). 54,59,61,62 PRE has been most successful in detecting long-range contacts in disordered protein ensembles. The method relies on the introduction of a paramagnetic nitroxide spin label at a specific position within the amino acid sequence.…”

“…Moreover, many examples in the literature have used PRE distance information to constrain MD simulations and finally arrive at a reasonably confined distribution of structural states in the ensemble of IDPs. 61,63 As compared to chemical shifts and NOEs, RDCs present a powerful tool that provides a far more extensive and quantitative description of local, and possibly, global order in the unfolded state. 55 Indeed, RDCs measured between pairs of nuclei in partially aligned proteins, are very sensitive probes of time and ensemble-averaged conformational equilibria exchanging on timescales up to the millisecond and can therefore be used to characterize both the structure and the dynamics of unfolded proteins.…”

From a structural average to the conformational ensemble of a DNA bulge

“…Compared with other NMR techniques, RDCs provide long-range angular structure information that helps to generate equilibrium ensemble models (9). In combination with molecular dynamic simulations, RDCs have been used to generate ensemble models for small disordered proteins (7,10), DNA duplexes (11), and a RNA bulge motif (12)(13)(14). In addition to RDCs, relaxation dispersion and paramagnetic relaxation enhancement have been used to detect and characterize conformational states that are in low abundance in an ensemble (5).…”